title: Dissection of the stepwise mechanism to beta-lactam formation and elucidation of a rate-determining conformational change in beta-lactam synthetase.
abstract: Clavulanic acid is a widely used beta-lactamase inhibitor whose key beta-lactam core is formed by beta-lactam synthetase. beta-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by beta-lactam formation. 32PPi-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 +/- 0.04), it was concluded that beta-lactam synthesis contributes at least partially to kcat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pKa approximately 8.1 apparent in the pH-kcat profile that stabilizes the beta-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on kcat attenuating the observed solvent isotope effect on beta-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 +/- 0.08 from a plot of log(o kcat/kcat) versus log(eta/eta(o)) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of > 1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 +/- 0.15) on kcat/Km. The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (DeltaH approximately 20 kcal/mol) comparable to the chemical barrier of beta-lactam formation.
authors.affiliation: Department of Chemistry and Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218.
authors.forename: Mary L
authors.initials: ML
authors.lastname: Raber
authors.affiliation: Department of Chemistry and Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Freeman
authors.affiliation: Department of Chemistry and Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218. Electronic address: ctownsend@jhu.edu.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Townsend
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D019818:Clavulanic Acid / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics
chemical_list: D004791:Enzyme Inhibitors; D019818:Clavulanic Acid; D000255:Adenosine Triphosphate; D000581:Amidohydrolases; C114020:beta-lactam synthetase